1. Field of the Invention
The present invention relates to a standard suspension used for inspection of liquids. More particularly, the invention relates to (1) a standard suspension that allows instruments that inspect and classify particle suspensions to be calibrated, validated, and/or compared and (2) a standard for the visual inspection of particle suspensions.
2. Description of Related Art
Biopharmaceutical companies often characterize biological drug products in injectable, liquid form in vials, syringes, or other containers. These containers are typically inspected for the presence of external particles but also can be inspected for particles formed by aggregation of the protein drug substance itself (e.g., in order to determine the safety of a drug product). Additionally, medical device manufacturers that coat medical devices with bio-compatible substances may inspect solutions after washing the devices to look for particles that have sloughed off the devices.
Similarly, in the field of Life Science, optical instruments such as microscopes and flow cytometers are used to analyze particles of biological nature, including biological cells, microvesicles, bacteria and viruses. These particles and protein aggregates, called biological particles henceforth, typically have a low refractive index that is close to that of the solution in which they are presented to the optical instrument.
Visual inspection by the human eye as well as various inspection instruments are used to detect biological particles. Results between human inspectors and instruments may, however, vary and the results may be difficult to compare given the random shapes and sizes of the biological particles and the relatively low contrast the biological particles exhibit because of their small refractive index difference with the solution in which they are suspended. FIG. 1 depicts a photograph of an example of protein aggregation in a container.
In order to assure the efficacy and safety of parenteral drugs, United States Pharmacopeia chapter <788> requires manufacturers of injectable drugs to report a count per unit volume of the number of any particles larger than 10 μm and a count per unit volume of the number of particles larger than 25 μm, including protein aggregate particles. Based on input from the new February 2013 United States FDA (Food & Drug Administration) guidelines that cite increasing concern about immunogenicity caused by protein aggregates, a new chapter with new rules may require monitoring biologic drug products for particulates greater than 2 μm. Protein aggregates may be difficult to detect because their index of refraction (n=˜1.34 to 1.40) is close to protein-based drug solutions index of refraction (n=˜1.34 to 1.36). There are also are a number of different protein agglomerate types that vary in shape and appearance. For example, from ellipsoidal to threadlike (aspect ratios from 0.8 to 0.1) and from amorphous to crystalline.
Current visual inspection relies on the transparency difference between the biologic particles and the surrounding liquid. Instruments used to inspect (detect) micron sized particles typically involve imaging, or light scattering, or light obscuration of the particles as they flow through a clear flow cell. The instruments may use visual and/or machine-based inspection processes. Each of these instruments is calibrated/validated for size measurement accuracy and particle count accuracy by the vendor or a user. Typically, calibration/validation is provided using polystyrene spheres (beads) of a well-defined and uniform diameter and defined concentration. Polystyrene beads are commercially available from vendors such as Thermo Fisher Scientific (Waltham, Mass., U.S.A.), Polysciences, Inc. (Warrington, Pa., U.S.A.), and other vendors.
There are three fundamental drawbacks to optical instrument calibration and validation using polystyrene beads. A first drawback is that polystyrene beads have a higher refractive index (typically n=1.599) than some particles of interest (e.g., protein aggregates with n=˜1.34 to 1.40). Thus, the optical signal of polystyrene beads does not adequately represent that of the signal generated by a low-contrast particle of the same size (such as biological particles). Biological particles may generate a lower signal than a polystyrene sphere of the same size due to the refractive index and transparency differences between the particles. As a result, the biological particle may be misclassified and result in erroneous analysis results. In addition, low contrast particle (e.g., biological particle) events may be eliminated from analysis when they generate a signal that falls below the analysis threshold established using polystyrene beads of the same size. This may result in erroneous count results of the low contrast particles. Likewise, the low refractive index makes biological particles more difficult to observe by human eye, also resulting in erroneous count results.
A second drawback is that some biological particles are irregular shaped three-dimensional (3-D) objects rather than uniformly spherical objects (like polystyrene beads). The irregular shapes of biological particles may present certain challenges to the optical analysis instruments. For example, the instrument may only generate a signal based on a two-dimensional (2-D) projection of the 3-D particle. Thus, measuring particles of irregular shape in a flow-through detector may produce wide distributions of instrument response due to random orientation of the particle at the interrogation point. Further, irregular shaped particles may result in a broader size distribution compared to the size distribution of polystyrene beads of the same size. As a result, irregularly shaped particles (such as some biological particles) may be eliminated from the analysis and result in erroneous count results.
A third drawback is that the concentration of polystyrene beads in suspension may vary due to systematic differences in sampling. For example, the parent polystyrene bead suspension may be sampled in the center of the suspension but after beads have begun to settle out of the suspension, resulting in different concentration in later samples.
Thus, it is desired to create a standard suspension that (1) allows the standardization of visual inspection methods and (2) allows optical analysis instruments to be calibrated, validated, and/or compared using standard particles that more closely match the properties of low-contrast and possibly irregular shaped particles such as biological particles. The standard particles in the standard suspension should be stable, reproducible, and have uniformity in key properties to allow operators to properly adjust the instruments or analysis software parameters when necessary. The particle standard suspension should support corrections to defined concentration through the use of an internal concentration standard. Proper adjustment of the instruments and/or the analysis software parameters may allow results to be compared accurately between samples, between analytical instruments, and/or between measurement labs.